Incorporation of Robust NIR‐II Fluorescence Brightness and Photothermal Performance in a Single Large π‐Conjugated Molecule for Phototheranostics

Abstract Second near‐infrared (NIR‐II, 1000–1700 nm) window fluorescence imaging‐guided photothermal therapy probes are promising for precise cancer phototheranostics. However, most of the currently reported probes do not demonstrate high NIR‐II fluorescent brightness (molar absorption coefficient (ε) × quantum yield (QY)) and photothermal performance (ε × photothermal conversion efficiency (PCE)) in a single molecule. Herein, a versatile strategy to solve this challenge is reported by fabricating a large π‐conjugated molecule (BNDI‐Me) with a rigid molecular skeleton and flexible side groups. The proposed BNDI‐Me nanoprobe boosts the ε and simultaneously optimizes its QY and PCE. Therefore, high NIR‐II fluorescent brightness (ε × QY = 2296 m −1 cm−1) and strong photothermal performance (ε × PCE = 82 000) are successfully incorporated in a single small molecule, and, to the best of knowledge, either of these two parameters is better than the best currently available fluorescent or photothermal probes. Thus, superior NIR‐II imaging effect in vivo and high photothermal tumor inhibition rate (81.2%) at low systemic injection doses are obtained. The work provides further insights into the relationship of photophysical mechanisms and structures, and presents promising molecular design guidelines for the integration of more efficient multiple theranostic functions in a single molecule.

operating at an acceleration voltage of 100 kV. Dynamic light scattering was performed on a particle size analyzer (NanoBrook 90Plus, Brookhaven Instruments Corporation). Absorption data was measured by Shimadzu UV-3600 ultraviolet-visible-near-infrared (UV-Vis-NIR) spectrophotometer. The NIR-II fluorescence spectra were monitored on a commercial NIR-II spectrophotometer (Fluorolog 3, Horiba) equipped with an 808 nm diode laser and an InGaAs NIR detector. The fs-TA spectra were performed with a Newport transient absorption spectrometer, in which a Spectra-physics Tsunami oscillator (800 nm) was used as the seed for a Spectra-Physics Spitre regenerative amplier (1 kHz, 7 mJ). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed by a PowerWave XS/XS2 microplate spectrophotometer (BioTek, Winooski, VT, USA). Confocal fluorescence imaging was conducted on ZEISS LSM880 laser scanning confocal microscope. Photothermal images were measured by a NIR thermal imager (FLIR E40), and the power density was measured by a VLP-2000 laser power meter. The in vivo and in vitro NIR-II fluorescence imaging
The flask was vacuumed and purged with dry nitrogen three times. Then anhydrous toluene (8 mL) was added, and the mixture was heated to reflux and stirred overnight in the dark. After cooling down to room temperature, the reaction mixture was extracted with DCM and water three times. The organic phase was combined, and dried with anhydrous Na 2 SO 4 . After removal of the solvent under reduced pressure, the residue was purified by column chromatography on silica gel using PE/DCM (v/v = 2:1 to 1:1) as the eluent to obtain BNDI-H (97 mg, 70%) as a dark brown solid. 1

Synthesis of Compound BNDI-Me
BNDI-H (80 mg, 0.038 mmol) and sodium hydride (60% in mineral oil, 30 mg, 0.76 mmol) were added into a 50 mL two-necked flask, then 10 mL dry THF was added, subsequently, the mixture was stirred at room temperature for 30 min under the protection of an argon atmosphere. Then, extreme excess of methyl iodide was added and the resulting mixture was heated to reflux with stirring. Reaction progress was monitored by thin-layer chromatography (TLC) analysis, and methyl iodide was added continuously. After completion, the THF was removed by rotary evaporation and obtained solid residue was extracted three times with DCM and water. The organic phase was dried over anhydrous Na 2 SO 4 , filtered, and the solvent was then removed by rotary evaporation. The crude product was purified using column chromatography on silica gel using a PE/DCM (v/v = 2:1 to 1:2) as eluent to yield a dark brown solid BNDI-Me (37mg, 45%). 1

DFT Calculations
Density functional theory (DFT) calculations were performed on Gaussian 09 program. The single molecular geometries and the frontier orbital energy levels were obtained by the Becke three-parameter hybrid functional combined with Becke-Lee-Yang-Parr correlation functional (B3LYP) with 6-31G(d) basis sets. Theoretical predictions on all long alkyl substituents were replaced with methyl groups in the calculations.

Preparation of nanoparticles (BNDI-H NPs and BNDI-Me NPs)
BNDI-H (1 mg) and F-127 (10 mg/mL) were dissolved in THF (2mL) by sonication. Then, mixed THF solution were used to prepare BNDI-H NPs by rapidly injecting them into distilled-deionized water (10 mL) under continuous sonication with a microtip-equipped probe sonicator (VCX 130, SONICS & MATERIALS, INC) for 2 min, the proportion of the ultrasonic power is set to 50%, and take ultrasound for 8 seconds and stop for 2 seconds as the time interval. After the ultrasound is over, the THF was blowed off with nitrogen under stirring at room temperature. Next, the aqueous solution was filtered through poly (ether sulfone) (PES) syringe driven filter (0.22 μm) (Millipore). Then the resulting solution was concentrated for further experiments. BNDI-Me NPs was prepared by the same method as above.

NIR-II fluorescence spectroscopy
The NIR-II fluorescence spectra were measured on a commercial NIR-II spectrophotometer (Fluorolog 3, Horiba). The detection wavelength range of the sample is in the 900 -1500 nm region when excited by 808 nm laser. The excitation laser beam is pass through the solution sample in a 1 cm path cuvette and the emission was collected with the transmission geometry.

Solvatochromic effect
In order to clearly understand the ICT effect, we first measured the absorption and emission spectra of BNDI-H and BNDI-Me in different polar solvents. And then this effect is evaluated quantitatively by the Lippert-Mataga equation: Where µg and µe are the dipole moment of ground state and excited state, respectively; and νabs and νem are the wavenumbers of the maximum absorption and fluorescence, respectively; h is the Planck constant, c is the light velocity, a is the radius of the Onsager cavity, and Δf can be calculated by following equation: where ε is the dielectric constant and n is the refractive index of the solvent. Ploting the Stokes shifts (νabs -νem) versus Δf to obtain the slope of the fitted line, and according to the value of h, c, a, the difference Δμ between the dipole moments of the excited state and the ground state can be calculated.

The measured method of the fluorescence quantum yield (QY)
A stock solution of IR-26 (the reference (QY = 0.5%)) in 1,2-dichloroethane was diluted to

Photothermal effect tests of BNDI-H NPs and BNDI-Me NPs
The where T max is the maximum stable-state temperature; T Surr is the surrounding temperature; h is the heat-transfer coefficient; s is the container's surface area; Q dis is the heat dissipation due to the light absorbing of container and water; A λ is the absorbance value of BNDI-H NPs at 808 nm; and I is the laser intensity (1 W/cm 2 ). The value of hs can be calculated by following on/off cycles to evaluate the photothermal stability, and IR-thermal camera was used to record the temperature curve during the thermal cycle.
The photothermal performance of BNDI-Me NPs was also measured by same method as above.

Cell culture
Hela and 4T1 tumour cells were incubated in Dulbecco's Modified Eagle Medium medium containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37 °C in humidified atmosphere of 5% CO 2 , and then the cells were collected for the following cell experiments.

Animal model
The 4T1-tumour-bearing mice were purchased from Jiangsu KeyGEN Biotech Corp., Ltd. and used in strict accordance with the guideline of the Laboratory Animal Center of Jiangsu KeyGEN Biotech Corp., Ltd., 4T1 cells (2 million) suspended in 50 mL of 50% v/v mixture of Matrigel in supplemented Dulbecco's modified Eagle's medium (10% fetal bovine serum, 1% pen/strep (100 IU/mL penicillin and 100 μg/mL streptomycin)) were injected subcutaneously in the right armpit of the mice to establish tumour models in 6-week-old female. Tumours were grown until a single aspect was ∼5 mm before being used for in vivo NIR-II fluorescence imaging experiment.

NIR-II Fluorescence Imaging of 4T1-Tumour-Bearing Mice
BNDI-Me NPs (100 μL, 0.5 mg/mL) were intravenously injected. NIR-II fluorescence imaging was explored at whole-body vasculature and tumours of living mice upon exposed to 808 nm laser (0.3 W/cm 2 ) irradiation, and signals were collected by a 1300 nm LP filter. And then, the mice were sacrificed, we further recorded the distribution of BNDI-Me NPs in the main organs, including tumour, heart, liver, spleen, lung and kidney.

In vitro photothermal therapy (PTT) efficacy
The MTT assay was used to determine in vitro cytotoxicity of BNDI-Me NPs in HeLa and The tumour volumes were measured and calculated based on the following formulae, e.i., tumour volume = (length × width 2 )/2. The mice were sacrifaced after 14 days, and the major organs (heart, liver, spleen, lung, kidney and tumour) were extracted and cell death and apoptosis levels were performed H&E staining and the TUNEL staining assay.

16.Statistical analysis
Data were presented as mean ± SD. The statistical significance of differences among the groups tested was determined using one-way ANOVA. **p < 0.01 was considered statistically significant.                          a) The molar extinction coefficients were directly obtained or calculated based on the data provided from absorbance spectra or references. b) The QY was re-calculated using QY of IR-26 = 0.5% in DCE as a standard. c) Absolute QY. d) The ε of cyanine in water is attenuated (~ 10 4 M -1 cm -1 ) than that in organic solution, generally, we choose ε of methanol with high polarity provided in the reference. e) IR-26 (Φ f = 0.1% in DCE) for BAF3-4 as reference. f) ICG (QY = 1%) in water were used as the reference for calculating the fluorescence QY of TA1 NPs. Figure S27. The chemical structure of organic molecules in Table S1.